The invention relates to the detection of ligands that cause conformational changes in their binding partners upon binding. As a result of the conformational change, electron transfer between two electron transfer moieties can occur.
There are a number of assays and sensors for the detection of the presence and/or concentration of specific substances in fluids and gases. Many of these rely on specific ligand/antiligand reactions as the mechanism of detection. That is, pairs of substances (i.e. the binding pairs or ligand/antiligands) are known to bind to each other, while binding little or not at all to other substances. This has been the focus of a number of techniques that utilize these binding pairs for the detection of the complexes. These generally are done by labeling one component of the complex in some way, so as to make the entire complex detectable, using, for example, radioisotopes, fluorescent and other optically active molecules, enzymes, etc.
Other assays rely on electronic signals for detection. Of particular interest are biosensors. At least two types of biosensors are known; enzyme-based or metabolic biosensors and binding or bioaffinity sensors. See for example U.S. Pat. Nos. 4,713,347; 5,192,507; 4,920,047; 3,873,267; and references disclosed therein. While some of these known sensors use alternating current (AC) techniques, these techniques are generally limited to the detection of differences in bulk (or dielectric) impedance, and rely on the use of mediators in solution to shuttle the charge to the electrode.
Recent work utilizes electron transfer for detection of nucleic acid and other analytes. See U.S. Pat. Nos. 5,824,473; 5,770,369; 5,591,578; 5,705,348; 5,780,234; PCT US98/12430; PCT US97/20014; PCT US95/14621; and PCT US98/12082.
The formation of specific protein-ligand complexes is often accompanied by large conformational changes (Uversky et al., Biochemistry (Moscow), 63:420-433, 1998), up to and including the ligand-induced folding of proteins unfolded under physiological conditions (reviewed in Plaxco and Gross, Nature, 386:657-659, 1997). In addition, a number of studies suggest that via deletions (e.g. Hamill et al., Biochemistry, 37:8071-8079, 1998; Flanagan et al., Proc. Natl. Acad. Sci. USA, 89:748-52, 1992) or core simplification (reviewed in Plaxco et al., Curr. Op. Struct. Biol., 8:80-85,1998; Desjarlis and Handel Prot. Sci., 4:2006-2018, 1995; Gassner et al., Proc. Natl. Acad. Sci. USA, 93:12155-12158, 1996) it may be possible to rationally engineer ligand-induced folding into naturally occurring or pahge-display generated (e.g. Vaughan et al., Nat. Biotechnol., 16:535-539, 1998; Wilson and Finlay, Can. J. Microbiol., 44:313-329, 1998) proteins featuring any arbitrary ligand specificity. These results suggest that an ability to conveniently monitor protein conformational changes could provide an important and commerically viable means of monitoring a wide variety of ligands of significant clinical, industrial, environmental or military interest.
While ligand-induced conformational changes could serve as an indicator of the presence or many specific ligands, the cumbersome, costly crystallographic or spectroscopic (reviewed in Plaxco and Dobson, Curr. Opin. Struc. Biol., 630-636, 1996) equipment required to monitor protein conformational changes has precluded the development of a viable ligand-detection technique based on this approach.
Electron transfer rates through biopolymers are extremely sensitive to donor-acceptor geometry, the structure and conformation of the intervening medium and the relative free and reorganization energies of the redox centers; see Casimiro et al., J. Phys. Chem., 97:13073-13077, 1993; Dutton and Mosser, Proc. Natl. Acad. Sci. USA, 91:10247-10250, 1994; Bjerrum et al., J. Bioenerg. Biomembr., 27:295-302, 1995; Langen et al., Science, 268:1733-1735, 1995; Daizadeh et al., Proc. Natl. Acad. Sci. USA, 94:3703-3708, 1998). In addition, the effects of protein conformational change on through-protein electron transfer rates has been reported; see Mutz et al., Proc. Natl. Acad. Sci. USA, 93:9521-9526,1996.
Accordingly, it is an object of the invention to provide for biosensors that can detect conformational changes that occur in proteins upon binding of ligands on the basis of electron transfer.